The botulinum neurotoxins (BoNTs) are the most poisonous substances known and have been classified by the CDC as one of the six highest-risk threat agents for bioterrorism. They are responsible for the disease botulism, a neuroparalytic condition that can progress to flaccid paralysis and death if left untreated. The BoNTs exist as one of seven immunologically distinct serotypes (A-G). They act by entering neurons and cleaving proteins that mediate the exocytosis of neurotransmitters. They are thought to bind neurons through a dual-receptor mechanism that includes interactions with both ganglioside and protein receptors. The identity of the protein receptor depends on which of the seven BoNT serotypes is present, but the molecular determinants of receptor specificity and dual-receptor binding are currently unknown. This proposal describes two specific aims that explore the structural and functional properties of BoNT-receptor interactions. Recent evidence suggests that BoNT/A changes conformation upon binding ganglioside and has different affinities for the three isoforms of the synaptic vesicle protein (SV2) receptor. In Aim 1, we will define the interactions between BoNT/A, ganglioside, and SV2 using X-ray crystallography, site-directed mutagenesis, and binding assays. The requirement for ganglioside may not be as stringent in the pathogenesis of the BoNT/B and BoNT/G serotypes. These BoNTs are highly similar but have notable differences in how they interact with the two isoforms of synaptotagmin (SytI and II). In Aim 2, we will use X-ray crystallography, site-directed mutagenesis and binding assays to delineate the specificity and affinity requirements for BoNT/G-SytI interactions. Comparison to BoNT/B-SytII interactions will provide a molecular answer to how BoNT/B and /G differ in their interactions with the two Syt isoforms, while simultaneously illuminating the common features that could be exploited for inhibiting receptor binding in both serotypes. Together, these studies will provide insight into the fundamental mechanisms by which BoNT gains access to neurons and generate new strategies for the design and improvement of anti-botulism therapeutics and preventatives. PUBLIC HEALTH RELEVANCE: Botulinum neurotoxin is a CDC category A toxin because it is easily produced and concocted into a bio- weapon for potential use in warfare and terrorism. In striking contrast to its destructive power, the potency of botulinum neurotoxin allows it to be used clinically in the treatment of dystonias, cerebral palsy, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's, and migraines. Understanding the molecular basis of how different forms of botulinum neurotoxin recognize neuronal cells will reveal important new insights into the pathogenic and therapeutic potential of this toxin.